Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

1/c expansion of a separable model of direct-interaction type

The properties of a proposed model of N point particles in direct interaction are considered in the limit of small velocities. It is shown that, in this limit, time correlations cancel out and that Newtonian dynamics is recovered for the system in a natural way.

1/c expansion of a separable model of direct-interaction type

The properties of a proposed model of N point particles in direct interaction are considered in the limit of small velocities. It is shown that, in this limit, time correlations cancel out and that Newtonian dynamics is recovered for the system in a natural way.

Hepatitis B and C virus coinfection: a novel model system reveals the absence of direct viral interference.

Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses, or viral interference. However, interactions between HBV and HCV have been difficult to study due to the lack of appropriate model systems. We have established a novel model system to investigate interactions between HBV and HCV. Stable Huh-7 cell lines inducibly replicating HBV were transfected with selectable HCV replicons or infected with cell culture-derived HCV. In this system, both viruses were found to replicate in the same cell without overt interference. Specific inhibition of one virus did not affect the replication and gene expression of the other. Furthermore, cells harboring replicating HBV could be infected with cell culture-derived HCV, arguing against superinfection exclusion. Finally, cells harboring replicating HBV supported efficient production of infectious HCV. Conclusion: HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in coinfected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV-HCV coinfection and may contribute to its clinical management in the future.

Mario Ruspoli et le "cinéma direct"

La production Argos Films édite chez Montparnasse le premier coffret DVD des principaux films du cinéaste. Profitant de cette accessibilité nouvelle, Décadrages a choisi de consacrer son 18e numéro à l'apport fondamental de Mario Ruspoli dans l'émergence, vers 1960, du cinéma documentaire en caméra légère, afin d'éclairer le contexte technique, discursif, théorique et artistique de cette production. Ce volume espère ainsi pallier cette méconnaissance de Mario Ruspoli en proposant, à travers cinq articles, la première publication universitaire sur ce cinéaste franco-italien.

La fi del censal: reducció de l'interès o vaga de pensions? (1750-1861)

La reducció de l'interès del 5 % al 3 % ha estat considerada el principi de la fi dels censals. Però aquest argument no té en compte que la de 1750 va ser només l'última d'una sèrie de reduccions del for de censal, que també es produïren a Anglaterra, França o Castella, i que els primers símptomes de retracció de l'oferta de préstecs censals no apareixerien fins a la darreria del segle XVIII o principi del segle XIX. L¿article proposa una explicació alternativa d¿aquelles reduccions, que ajustaven l'interès a l'alça coetània del valor patrimonial de la terra i la minva de la rendibilitat del seu esmerç, i de la fi del censal a partir de l'impagament de pensions que es desencadenà a les acaballes del segle XVIII, juntament amb la vaga de delmes i rendes de la terra, quan s'havien esgotat, en bona part, les possibilitats d'expansió agrària del segle XVIII.

A simple blood-culture bacterial pellet preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing with the VITEK 2 system.

An ammonium chloride procedure was used to prepare a bacterial pellet from positive blood cultures, which was used for direct inoculation of VITEK 2 cards. Correct identification reached 99% for Enterobacteriaceae and 74% for staphylococci. For antibiotic susceptibility testing, very major and major errors were 0.1 and 0.3% for Enterobacteriaceae, and 0.7 and 0.1% for staphylococci, respectively. Thus, bacterial pellets prepared with ammonium chloride allow direct inoculation of VITEK cards with excellent accuracy for Enterobacteriaceae and a lower accuracy for staphylococci.

Direct Care Worker Compensation Advisory Committee Review and Recommendation Report, December 11, 2008

The 2008 general assembly acknowledged in House File 2539, Section 70 that is recognizes direct care workers play a vital role and make a valuable contribution to Iowa's Health Care Reform efforts in providing care to Iowans with a variety of needs in both institutional and home and community based settings. the legislation identified that recruiting and retaining highly competent direct care workers is a challenge across all healthcare employment settings.

Report to the Governor and General Assembly on Voluntary Employer-Sponsored Health Care Coverage Demonstration Project-Non-licensed Direct Care Workers, December 2008

Iowa faces a growing crisis in providing an adequate number of direct care workers for its again population. Direct care workers caring for the aging and disabled population are among those in our work force with the lowest wages and lack of access to health insurance. A survey of direct care workers conducted by the Iowa Caregivers Association in June of 2066 indicated that benefits ranked second only to wages in the reasons for job-hoping. A study of the direct care workforce in Vermont healthy insurance, ranked second only to wages, as important to attracting and keeping direct care workers.

A two-level analysis of the determinants of direct democratic choices in European, immigration and foreign policy in Switzerland

This article employs a unique data set - covering 25 popular votes on foreign, European and immigration/asylum policy held between 1992 and 2006 in Switzerland - in order to examine the conditional impact of context upon utilitarian, cultural, political and cognitive determinants of individual attitudes toward international openness. Our results reveal clear patterns of cross-level interactions between individual determinants and the project-related context of the vote. Thus, although party cues and political competence have a strong impact on individuals' support for international openness, this impact is substantially mediated by the type of coalition that is operating within the party elite. Similarly, subjective utilitarian and cultural considerations influence the voters' decision in interaction with the content of the proposal submitted to the voters as well as with the framing of the voting campaign.

Application of direct-infusion ESI-MS/MS for toxicological screening.

BACKGROUND: Direct-infusion ESI-MS/MS is a powerful approach for the identification of substances in complex mixtures. The aim of this work was to investigate its applicability to the toxicological screening of blood samples. A simple protein precipitation was used, followed by a 15 min infusion of the extract in the mass spectrometer. RESULTS: The application of the procedure to commercial quality controls and authentic post-mortem blood samples demonstrated that the direct-infusion ESI-MS/MS approach enables the simultaneous identification of substances that require different chromatographic conditions. However, poor sensitivity was observed for benzodiazepine, amphetamines and opiate compounds. CONCLUSION: Considering the facile implementation and positive performance of direct-infusion ESI-MS/MS, this approach may to be a valuable complementary technique for systematic toxicological analysis procedures.